1. Field of the Invention
The present invention relates to antibodies to murine Tissue factor (mTF) that bind to one or more epitopes in the amino acid sequences of mTF that are in the same region as the epitope or epitopes to which the antibody 5G9 binds in human Tissue Factor and thus have properties similar to the 5G9 antibody. The murine antibodies of the invention are useful as research tools for evaluating the therapeutic potential of anti-tissue factor antibodies that neutralize TF activity by inhibiting the activation of Factor X and for exploring the role of TF in various biological processes.
2. Background
The coagulation of blood involves a cascading series of reactions leading to the formation of fibrin. The coagulation cascade consists of two overlapping pathways, both of which are required for hemostasis. The intrinsic pathway comprises protein factors present in circulating blood, while the extrinsic pathway requires tissue factor (TF), which is expressed on the cell surface of a variety of tissues in response to vascular injury (Davie et al., 1991, Biochemistry 30:10363). When exposed to blood, TF sets in motion a rapid cascade of activation steps that result in the formation of an insoluble fibrin clot (See FIG. 1).
TF has been investigated as a target for anticoagulant therapy. TF (also known as thromboplastin, CD142 and coagulation factor III) is a single chain, 263 amino acid membrane glycoprotein that functions as a receptor for factor VII and VIIa and thereby initiates the extrinsic pathway of the coagulation cascade in response to vascular injury. TF is an integral membrane protein normally present on the cell surface of non-vascular cell types. Healthy endothelial cells lining normal blood vessels do not produce TF, however, TF is always present in the adventitia of blood vessels.
TF serves as both a cofactor for factor VIIa, forming a proteolytically active TF:VIIa complex on cell surfaces, and as a Vila receptor, inducing downstream intracellular changes (Bazan, J F, Proc. Natl. Acad. Sci USA (1990) 87:6934-8; Reviewed by Konigsberg, et al. Thromb. Haemost. (2001) 86:757-71). In addition to its role in the maintenance of hemostasis by initiation of blood clotting, TF has been implicated in pathogenic conditions. Specifically, the synthesis and cell surface expression of TF has been implicated in vascular disease (Wilcox et al, 1989, Proc. Natl. Acad. Sci. 86:2839) and gram-negative septic shock (Warr et al., 1990, Blood 75:1481). Furthermore, in a number of pathological states involving an acute inflammatory response and progression to a thrombotic state, such as sepsis, increased TF expression on the vascular endothelium results from the release of inflammatory mediators such as s TNF and/or IL-1.
The Role of TF in Cancer
Tissue factor is also overexpressed on a variety of malignant tumors and isolated human tumor cell lines, suggesting a role in tumor growth and survival. TF is not produced by healthy endothelial cells lining normal blood vessels but is expressed on these cells in tumor vessels. It appears to play a role in both vasculogenesis in the developing animal and angiogenesis in normal and malignant adult tissues. Inhibition or targeting of TF may therefore be a useful anti-tumor strategy that could affect the survival of TF overexpressing tumor cells directly by inhibiting TF mediated cellular signaling or other activities. In addition, this approach may prevent tumor growth indirectly via an antiangiogenic mechanism by inhibiting the growth or function of TF expressing intra-tumoral endothelial cells.
WO94/05328 discloses the use of anti-TF antibodies to inhibit the onset and progression of metastasis by abolishing the prolonged adherence of metastazing cells in the microvasculature thereby inhibiting metastasis, but does not disclose any effect on the growth of established tumor cells.
The Role of TF in Transplantation
Despite the central role of TF in blood coagulation, the mechanisms underlying the regulation of TF pro-coagulant activity in vivo are still being explored as are non-coagulant activities related to receptor signaling (Morrissey, J. H. Thromb Haemost 2001; 86:66-74 and Key, N. S., Bach, R. R. Thromb Haemost 2001; 85:375-6).
Unperturbed cells in culture have weak coagulant activity, however, cells or tissues that have been disrupted or stimulated with e.g. growth factors or endotoxin leading to increased intracellular calcium ion (Ca++) display fully expressed and active TF. Perturbation of the phospholipid species between the inner and outer cell membrane leaflets, especially phosphatidyl serine, was implicated as a possible trigger of this de-encryption of a macromolecular substrate binding site on TF which defines TF activation (Bach, R. R, Moldow, C. F. Blood 1997; 89 (9): 3270-3276).
A number of reports implicate the role of active TF in the pathogenesis of transplant failure. U.S. Pat. No. 6,387,366 notes that bone marrow stem cell (BMSC) transplantation causes blood clotting or hemorrhage due to the expression of TF on the infused cells and suggests several methods to reduce the biological activity of TF or FVII in infusions employing BMSC transplantation, gene therapies employing BMSC, and other types of cell transplantation. These methods include treating the preparation or the patient with TF antagonists.
TF Antagonists
Various anti-TF antibodies are known. For example, Carson et al, Blood 70:490-493 (1987) discloses a monoclonal antibody prepared from hybridomas produced by immunizing mice with human TF purified by affinity chromatography on immobilized factor VII. Ruf et al, (1991, Thrombosis and Haemostasis 66:529) characterized the anticoagulant potential of murine monoclonal antibodies against human TF. The inhibition of TF function by most of the monoclonal antibodies that were assessed was dependent upon blocking the formation or causing the dissociation of the TF/VIIa complex that is rapidly formed when TF contacts plasma. Such antibodies were thus relatively slow inhibitors of TF in plasma as factor VII/VIIa remains active. One monoclonal antibody, TF8-5G9, was capable of inhibiting the TF/VIIa complex by blocking the F.X binding site without dissociating the complex, thus providing an immediate anticoagulant effect in plasma which is not absolute as F.VII is still available (See FIG. 1). This antibody is disclosed in U.S. Pat. Nos. 6,001,978, 5,223,427, and 5,110,730. Ruf et al. suggest that mechanisms that inactivate the TF/VIIa complex, rather than prevent its formation, may provide strategies for interruption of coagulation in vivo. In contrast to other antibodies that inhibit factor VII binding to TF, TF8-5G9 shows only subtle and indirect effects on factor VII or factor Vila binding to the receptor. TF8-5G9 binds to defined residues of the extracellular domain of TF that are also involved in F.X binding with a nanomolar-binding constant (See FIG. 2) Thus, TF8-5G9 is able to effectively block the subsequent critical step in the coagulation cascade, the formation of the TF:VIIa:X ternary initiation complex (Huang et al, J. Mol. Biol. 275:873-894 1998).
Anti-TF monoclonal antibodies have been shown to inhibit TF activity in various species (Morrissey et al, Throm. Res. 52:247-260 1988) and neutralizing anti-TF antibodies have been shown to prevent death in a baboon model of sepsis (Taylor et al, Circ. Shock, 33:127 (1991)), and attenuate endotoxin induced DIC in rabbits (Warr et al, Blood 75:1481 (1990))
WO 96/40921 discloses CDR-grafted anti-TF antibodies derived from the TF8-5G9 antibody. Other humanized or human anti-TF antibodies are disclosed in Presta et al, Thromb Haemost 85:379-389 (2001), EP1069185, U.S. Pat. No. 6,555,319, WO 01/70984 and WO03/029295.
An antibody that specifically recognizes TF and inhibits coagulation may provide a useful therapy for diseases where thrombogenesis is abnormal. However, to evaluate the potential efficacy of an anti-TF antibody in vivo, the antibody must cross react with TF from the animal or a surrogate must be identified that acts in a similar manner to the anti-human TF antibody. In vitro experiments have demonstrated that the anti-human TF antibody, 5G9, does not bind to murine TF. This observation is consistent with the structural data and with the differences in the mouse and human proteins in the region that constitutes the 5G9 epitope. Indeed, there are eight residues within the epitope that are different between murine and human TF (FIG. 3). Efforts to generate an anti-murine TF antibody that acts in a manner similar to 5G9 by immunization of rats or other animals have not heretofore been successful.
Phage display technology describes an in vitro selection technique in which the polynucleotide sequence encoding a peptide or protein is genetically fused to a coat protein of a bacteriophage, resulting in display of the fused protein on the exterior of the phage virion, while the DNA encoding the fusion resides within the virion. This physical linkage between the displayed protein and the DNA encoding it allows screening of vast numbers of variants of the protein, each linked to its corresponding DNA sequence, by a simple in vitro selection procedure called “biopanning”.
Phage displayed antibody libraries have become a valuable tool for generating human antibodies or antibodies with selected specificities (Hogenboom, H. et al. 2000. Immunology Today 21(8), 371-378). Domain directed pannings have become a routine way of selecting antibodies that bind to a variety of epitopes on a target protein.
Ligand-capture directed panning is analogous to an ELISA sandwich assay in that an immobilized antibody to an irrelevant and non-adjacent epitope is used to capture and present the preferred binding face of the target ligand for phage panning (U.S. Pat. No. 6,376,170). Others have used competing antibodies to selectively mask the antigen at other than the desired target domain (Tsui, P. et al. 2002. J. Immunol. Meth. 263:123-132). Pathfinder technology uses a monoclonal and polyclonal antibodies, as well as natural ligands conjugated directly or indirectly to horseradish peroxidase (HRP). In the presence of biotin tyramine these molecules catalyze biotinylation of phage binding in close proximity to the target antigen, allowing specific recovery of ‘tagged’ phage from the total population using streptavidin. In this way, phage binding to the target itself, or in its immediate proximity, are selectively recovered (Osborn, J. K. et al. 1998. Immunotechnol. 3: 293-302). The use of monoclonal antibodies to direct binding to alternate sites has also been termed “epitope walking” (Osborn, J. K. et al. 1998. supra).
Such selections have primarily been achieved by employing a stepwise selection of antibodies. In the first stage of selection, a variety of antibodies are selected to the target protein. In the second stage of selection, panning is performed in the presence of one or more selected antibodies so that any newly selected antibodies must bind at a different epitope. The present invention employed a unique methodology that incorporates a hybrid human:murine TF competitor or decoy protein in the panning process to select antibodies that bind to the same regions as the 5G9 epitope. Several antibodies were selected that specifically interact with murine TF but not the hybrid protein and have properties similar to the parent anti-human TF antibody.